The miScript Single Cell qPCR Kit includes reagents for cell lysis, cDNA synthesis and unbiased preamplification of all microRNAs (miRNAs), facilitating the expression analysis of miRNAs from individual cells. One to 100 cells or 25 pg to 1 ng of purified RNA is required as template for each miScript Single Cell qPCR Kit reaction. Unlike other single-cell miRNA expression products, the miScript Single Cell qPCR Kit is a completely universal kit. Starting from a single cell, all miRNAs, both annotated and non-annotated, are selectively converted into cDNA and subsequently amplified. There is no fractionation of the sample or selection of specific miRNAs required. From cell lysis through preamplification, the miScript Single Cell qPCR Kit is a robust, universal solution for precision single-cell miRNA expression. An entire miRNome expression profile can be generated from an individual cell – explore the power of one.

Why is single cell analysis important?

Tissues are heterogeneous mixtures of different cell populations with each cell contributing a unique proteome and transcriptome. For example, normal and disease-biology are both inherently heterogeneous, and cells can respond individually and in concert to internal and external stimuli. Further, individual cells can differ due to epigenetics, circadian clock, cell cycle, microenvironments, cell-to-cell contacts and intrinsic transcriptional “noise.” While bulk transcriptomic analysis of mRNA, lncRNA and miRNA expression is critical for understanding biological systems, the consequential “cellular averages” mask intrinsic transcriptional variability across individual cell subpopulations. In fact, the contribution of rare cell subtypes may be completely obscured when cells are assessed in bulk. Single-cell expression analysis brings into focus the individual contribution of every cell, providing a complete, granular understanding of a specific biological response.

The miScript Single Cell qPCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Principle

Mature miRNAs are naturally occurring, 22-nucleotide, non-coding RNAs that mediate posttranscriptional gene regulation. Animal mature miRNAs possess a 3’ hydroxyl group and a 5’ phosphate group (unlike most other cellular RNAs). Due to this, adapters can be ligated to both the 3’ end and 5’ end of mature miRNAs, enabling universal reverse-transcription and preamplification, while minimizing the background from other RNA species. The miScript Single Cell qPCR Kit includes all of the reagents required to perform cell lysis through preamplification in a streamlined, integrated workflow.

Procedure

With the miScript Single Cell qPCR Kit, you can go from a single cell or minute amounts of purified RNA to isolated and amplified miRNAs in a single, streamlined workflow. There is no fractionation of the sample or selection of specific miRNAs required. The kit integrates with our miScript miRNA PCR Arrays and miScript Primer Assay to allow you to reliably perform whole miRNome profiling, disease/pathway-specific miRNA expression analysis, or individual miRNA quantification from a single cell.
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Real-time PCR for the Fluidigm BioMark System

Preamplified product from the miScript Single Cell qPCR Kit can serve as the template for real-time PCR on the Fluidigm BioMark System using either miScript miRNA PCR Arrays or miScript Primer Assays and the miScript Microfluidics PCR Kit. This specialized real-time PCR kit contains the miScript Microfluidics Universal Primer (reverse primer) and Microfluidics qPCR Master Mix (specifically optimized for the Fluidigm BioMark System; contains EvaGreen dye for detection and ROX passive reference dye). For more information, please refer to the miScript Microfluidics Handbook.

miScript Primer Assays: Preamplified product from the miScript Single Cell qPCR Kit serves as the template for real-time PCR analysis using a miScript Primer Assay and the miScript SYBR Green Kit. For more information, please refer to the miScript PCR System Handbook.